ENVIRONMENTAL PROTECTION AGENCY 40 CFR Parts 110 and 300 National Oil and Hazardous Substan ces Pollution Contingency Plan U.S. Environmental Protection Agency (EPA) Proposed rule. Acute Toxicity Testing Protocol Including Procedures for Product/Oil Mixtures The Agency is proposing provisions for conducting product

- only acute toxicity tests with all agent categories (i.e., dispersants, surface washing agents, bioremediation agents, solidifiers, and herding agents). However, the Agency is proposing provisions for conducting acute toxicity tests with product

- oil mixtures for dispersants only, and not for the other agent categories. EPA believes that non

- dispersant products are not likely to be used in the same quantities or durations as dispersants a nd further, that some may be recovered. Thus, making a requirement to conduct toxicity tests of product/oil mixtures for agent categories other than dispersants may not be necessary. There may be concerns, however, for increased product toxicity when no n - dispersant products interact with the oil. Thus, the Agency is requesting comment on the need, in addition to the proposed acute toxicity testing requirements for surface washing agents, bioremediation agents, solidifiers, and herding agents, to include requirements for product

- reference oil mixtures for these categories. A protocol that includes procedures for the preparation of product/oil mixtures for toxicity testing of these other agent categories is provided below for review as an alternative to the one included in the proposed rule. This revised protocol is consistent with the testing protocol proposed for dispersants . The Agency is requesting comments on the protocol itself, including the preparation procedures for the product

- oil mixtures

. The redline d text indicates changes to the proposed protocol that include the preparation of prod uct/oil mixtures for toxicity testing. Specifically, the Agency is requesting comment on whether it should require testing of alternative product to oil ratio s for non

- dispersant products to reflect potentially higher product concentrations that may be recommended by the manufacturer.4. 0 S tandard Acute Toxicity Testing of Surface Washing Agents, Bioremediation Agents, Herding Agents, and Solidifiers.
4.1 Summary. This laboratory protocol includes testing for: 1) saltwater standard static acute toxicity tests for test products with the mys id shrimp , Americamysis bahia (48

- hr duration) and the inland silverside, Menidia beryllina (96

- hr duration); 2) test product/oil mixture static acute toxicity tests for Americamysis bahia and Menidia beryllina (48

- hr and 96

- hr duration, respectively); 3 ) freshwater standard static acute toxicity tests for test products with the daphnid , Ceriodaphnia dubia (48

- hr duration) and the fathead minnow, Pimephales promelas (96

- hr duration ); and 4) test product/oil mixture static acute toxicity tests for Ceriodaphn ia dubia and Pimephales promela s (48

- hr and 96

- hr duration, respectively) ( see Table 8 of this Appendix ).
TABLE 8

– TOXICITY TESTING REQUIREMENTS FOR SURFACE WASHING AGENTS, HERDING AGENTS, BIOREMEDIATION AGENTS AND SOLIDFIERS Test Procedure
Application Environment 96
- hr Static Acute: Menidia beryllina 48
- hr Static Acute: Americamysis bahia 96
- hr Static Acute: Pimephales promelas 48
- hr Static Acute: Ceriodaphnia dubia Saltwater only yes yes no no Freshwater only no no yes yes Freshwater and saltwater use yes yes yes yes

4.2 Dilution Water . Use Section 7 of EPA’s Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA

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4.3 Preparation of Stock Solutions

. 4.3.1 Liquid Surface Washing Agents an d or Herding Agents . Prepare a 1000 μL/L stock solution prior to test initiation by adding 1.1 mL of test product to 1100 mL of dilution water in aglass vessel. Place on a magnetic stir plate then add and center a stir bar and adjust the stir plate to ob tain a vortex of 25% of the total volume of the liquid. Mix the resulting stock solution for approximately five minutes at room temperature. Using a glass pipette, remove appropriate aliquots of stock solution from between the mixing vessel wall and edge of the vortex and place directly into the dilution water within an exposure vessel. Base the preparation of exposure solutions on the nominal concentration of the stock solution and follow procedures outlined in

s ections 4.6 and/or 4.7 of this Appendix , as appropriate.
4.3.2 Bioremediation Agents . For products consisting of two or more liquid and/or solid components, prepare the product following the manufacturers recommended procedure and ensure the test product mixture is completely blended. Prepare a 1000 μL/L stock solution prior to test init iation by adding 1.1 mL of the test product mixture to 1100 mL of dilution water in a glass vessel. Place on a magnetic stir plate then add and center a stir bar and adjust the stir plate to obtain a vortex of 25% of the total volume of the liquid. Mix t he resulting stock solution for approximately five minutes at room temperature. Using a glass pipette, remove appropriate aliquots of stock solution from between the mixing vessel wall and edge of the vortex and place directly into the dilution water withi n an exposure vessel. Base the preparation of exposure solutions on the nominal concentration of the stock solution and follow procedures outlined in

s ections 4. 6 and/or 4. 7 of this Appendix , as appropriate.
4.3.3 Solid Phase Products . Assessment of the t oxicity of solidifiers and other solid phase products are determined using the aqueous phase of water

- accommodated fractions (WAFs) of the test product. Fit a glass aspirator bottle (approximately 23L) equipped with hose bib at the base with a length of s ilicon tubing containing a hose clamp. Fill the bottle with 19L of dilution water leaving a 20% headspace above the liquid, place on a magnetic stir plate then add andcenter a stir bar. Add the test product at 25 g/L and securely seal the bottle using a silicon stopper and wraps of parafilm. Adjust the stir plate to obtain a vortex of 25% of the total fluid volume, stir for 18 hours then settle for 6 hours. Maintain the temperature at 25 0 C during stirring and settling. Purge the hose at the base of the bottle of any material followed by removal of the WAF (aqueous phase) into a clean glass container without disturbing the product on the surface. The WAF should be remixed and used for the preparation of exposure solutions following procedures outlined i n s ection 4.4 of this Appendix

. 4.3.4 Test Product

- Reference oil(s) Mixtures . Use IFO 120 and non

- weathered ANS oils. To obtain these oils, see http://www.epa.gov/emergencies/content/ncp /index.htm . Assessment of test product

- reference oil mixture toxicity is determined for each reference oil using the aqueous phase of a chemically enhanced

- water accommodated fraction (CE

- WAF). Repeat the following procedure for each reference oil tested. Fit a gla ss aspirator bottle (approximately 23L) equipped with hose bib at the base with a length of silicon tubing containing a hose clamp. Fill the bottle with 19L of seawater or freshwater , as appropriate, leaving a 20% headspace above the liquid, place on a ma gnetic stir plate then add and center a stir bar. Add the respective crude oil at 25 g/L using a silicon tube attached to a glass funnel that reaches just below the water surface. Using this method reduces the production of air bubbles on the oil surface slick. Adjust the stir plate to obtain an oil vortex of 25% of the total volume of the seawater or freshwater, as appropriate, then add the product to be tested at a ratio of 1:10 product: oil (2.5 g/L). Securely seal the bottle to reduce the loss of vola tiles using a silicon stopper and wraps of Parafilm and stir for 18 hours, then allow the solution to settle for 6 hours. Maintain the temperature at 25 0 C during stirring and settling. Purge the hose at the base of the bottle of any material followed by removal of the CE

- WAF (aqueous phase) into a clean glass container without disturbing thesurface oil slick. The CE

- WAF should be remixed and 1

- 2 L removed for chemical analysis of TPH following the procedures outlined in Section 4.5. The remaining volum e will be used for the preparation of exposure solutions following procedures outlined in Section 4.4. To reduce time and cost, it is preferable to mix sufficient amounts of test product

- reference oil mixture CE

WAF to allow preparation of exposure soluti ons for conducting simultaneous acute tests with the two saltwater species and/or the two freshwater species, as appropriate

. 4.4 Preparation of Exposure Concentrations

. 4.4.1 Concentration Selection . Preliminary rangefinder tests may be necessary using a series of logarithmic concentrations (e.g. 0.1, 1, 10, 100 μl test product/L or mg TPH/L ) to determine the appropriate exposure concentration range necessary to determine LC50 values and 95% confidence intervals. For definitive tests, conduct a minimum of five test concentrations using a geometric ratio between 1.5 and 2.0 (e.g. 2, 4, 8, 16, and 32). Note that when testing only the test product, the highest test concentration should not exceed the test products self

dispersibility limi t , with exception of solid phase products

. 4.4.2 Exposure Concentrations . Exposure solutions are prepared by adding the appropriate amount of stock solution directly to dilution water in each test chamber. Mix each exposure solution using five rotations in one direction followed by five rotations in the opposite direction using a solid glass stir rod.
4.4.3 Reference Toxicants . Separate toxicity tests must be performed with a reference toxicant for each species tested. Conduct additional reference toxic ity tests any time a change in the culture population or source of a test species occurs. Use reagent grade quality sodium dodecyl sulfate (SDS), also known as dodecyl sodium sulfate (DSS), and sodium lauryl sulfate (SLS) as the reference toxicant. Informa tion on procedures for conducting reference toxicanttests with these species can be found in s ection 4 of EPA’s Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA

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. 4.5 Chemical Analysis of Stock Solutions . Add the 1L sample of CE

- WAF (Section 4.3.d) solutions directly to amber glass bottles with Teflon ®

- lined cap. Collect a replicate sample in the event of accidental loss or if reanalysis of the stock solution becomes necessary. Adjust sample to a pH=2 using 50% hydrochloric acid, immediately refrigerate and analyze within 48 hours of collection. Analyze samples for C9

- C32 total petroleum hydrocarbons (TPH) by gas chromatography

- flame ionization detection (GC

- FID) fol lowing EPA SW

- 846, Method 8015B

DRO. Report TPH concentration of stock solutions as milligrams TPH/L and use in the calculation of exposure concentrations for all toxicity tests conducted with CE

- WAF.
4.6 Saltwater Static Acute Tests with Menidia beryllin a and Americamysis bahia
4.6.1 General . Use EPA’s Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA

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- 012) [1] for testing each species separately with the test product or a mixture of the test product and reference oil

. 4.6.2 Test Solutions . Modify procedures in EPA

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- 012 specifically dealing with the handling and toxicity testing of effluents or receiving water samples as follows: Prepare stock solutions following the appropriate sections (4.3. 1 , 4.3. 2 , or 4.3. 3, and 4.3.4 ) and exposure concentrations following s ection 4.4 of this A ppendix

. 4.6.3 Number of Treatments, Replicates and Organisms. Conduct a minimum of three replicates of at least five exposure treatments p lus a minimum of three replicate dilution water controls. Expose ten organisms per replicate treatment.4.6.4 Exposure Period. Test duration is 48

- hr for A. bahia and 96

- hr for M. beryllina

. Mortality must be recorded at each 24 hour period of each test.
4.6.5 Test Acceptability. For each test performed, survival of control animals must be > 90% and test results must allow determination of statistically valid LC50 and 95% confidence interval values except in cases where the LC50 is > 1000 μl/L or is determined to be greater than the limits of water solubility of dispersibility.
4.6.6 Static Acute Test Summary . A summary of required test conditions is provided in Table 9

. 4. 7 Freshwater Static Acute Tests with Pimephales promelas and Ceriodaphnia dubia
4.7.1 General . Use EPA’s Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA

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- 012) [1] for testing each species separately with the test product or a mixture of the test product and reference oil

. 4.7.2 Test Solutions . Modify procedures in EPA

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- 012 specifically dealing with the handling and toxicity testing of effluents or receiving water samples as follows: Prepare stock solutions following the appropriate sections (4.3. 1 , 4.3. 2 , or 4.3. 3, and 4.3.4 ) and exposure concentrations following s ection 4 .4 of this A ppendix

. 4.7.3 Number of Treatments, Replicates and Organisms. P. promelas : Conduct a minimum of three replicates of at least five exposure treatments plus a minimum of three replicate dilution water controls. Expose ten organisms per replicate treatment. C. dubia : Conduct a minimum of four replicates of at least five exposure t reatments plus a minimum of four replicate dilution water controls. Expose five organisms per replicate treatment.
4.7.4 Exposure Period. Test duration is 48

- hr for C. dubia and 96

- hr for P. promelas

. Mortality must be recorded at each 24 hour period of each test.4.7.5 Test Acceptability. For each test performed, survival of control animals must be > 90% and test results must allow determination of statistically valid LC50 and 95% confidence interval values except in cases where the LC50 is >1000 μl/L o r is determined to be greater than the limits of water solubility of dispersibility.
4.7.6 Static Acute Test Summary . A summary of required test conditions is provided in Table 9

. 4. 8. Laboratory Report . The laboratory must include, for each toxicity test report, all applicable information, data and analyses as follow : 4.8.1 Test Objective : protocol title and source, endpoint(s);
4.8.2 Product Information : product name, manufacturer contact informatio n, lot number, production date, date received / chain of custody;
4.8.3 Contract Facility : contact information;
4.8.4 Dilution Water: source, pretreatment, physical and chemical characteristics (pH, salinity);
4.8.5 Test Conditions: date and time of te st (start and end), test chambers type and volume, volume of solution per chamber, number of organisms per chamber, number of replicate chambers per treatment, feeding frequency, amount and type of food, test concentrations, test temperature (mean and rang e), test salinity (mean and range);
4.8.6 Test Organisms: common and scientific name, source contact information, age and date purchased, acclimation conditions (e.g., temperature, salinity, both mean and range), age at
test start; 4.8.7 Reference toxica nt: date received, lot number, date of most recent test, results and current Cumulative Sum Chart, dilution water used, physical and chemical methods used;4.8.8 Quality Assurance: verification of laboratory accreditation, including subcontractor faciliti es; 4.8.9 Test Results: raw data in tabular and graphical form, daily records of affected organisms in each concentration replicate and controls, table of required endpoints (i.e., LC50, 95% CI, IC50 , LOEC and NOEC ), statistical methods used to calculate endpoints, summary tables of test conditions and QA data;
4.8.10 Analytical Results : method summary including Limit of Detection (LOD)/Limit of Quantitation (LOQ), deviations and reasons if any, sample summary, results including chromatograms and data qualifiers, QA summary including calibration curves, method blank and surrogate recover y, analytical results summary; and
4.8.11 Conclusions: Relationship between test endpoints and threshold limit.TABLE 9

– SUMMARY OF TEST CONDITIONS Saltwater Acute M. beryllina Saltwater Acute A. bahia Freshwater Acute P. promelas Freshwater Acute C. dubia Test type Static non

renewal Static non

renewal Static non

renewal Static non

renewal Test duration 96 hours 48 hours 96 hours 48 hours Salinity 20 ± 2‰ 20 ± 2‰ NA NA Temperature 25 ± 1 0 C (recommended) . Test temperatures must not deviate from 25 0 C (maximum minus minimum temperature) by more than 3 0 C during the test (required). Light quality Ambient laboratory illumination Light intensity 10

- 20 μE/m 2 /s Photoperiod 16 h light, 8 h darkness, with phase in/out period recommended Test chamber size 1 250 mL 250 mL 250 mL 30 mL Test solution volume 1 200 mL 200 mL 200 mL 15 mL Age of test organism 2 9

- 14 days 1

- 5 days 1

- 14 days <24 hours No. organisms per test chamber 10 10 10 5 No. of replicate chambers per concentration (minimum) 3 3 3 4 Feeding regime Refer to specific feeding procedures provided in each test method Aeration None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate :< 100 bubbles/minute Test concentrations 5 exposure concentrations and a control (minimum required) Test acceptability (required) ≥90% survival in controls
1 Recommended minimum value. 2 Less than or equal to 24

- hr range in age. 4.9 References for Section 4 (1) U.S. EPA. 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. U.S. Environmental Protection Agency, Washington, D C (EPA 821

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- 012).